A number of procedures exist for the preparation of competent cells and the introduction of DNA into these cells. For example Mandel and Higa (Journal of Molecular Biology 53:159 (1970)) describe a procedure whereby bacteriophage DNA is combined with E. coli cells in the presence of 50 mM Ca++at 0° C., followed by a brief heat pulse at 37° C.-42° C. This method has been extended to the uptake of chromosomal DNA (Cosloy and Oishi, Proceedings of the National Academy of Science 70:84 (1973)) and plasmid DNA (Cohen et al., Proc. Natl. Acad. Sci. USA 69:2110 (1972)). A summary of the factors influencing the efficiency of transformation is given in Hanahan (JMB 166:557 (1983)). These factors include the addition of other cations such as Mg, Mn, or Rb to the Ca-treated cells as well as the prolonged incubation of the cells in CaCl2. The efficiency of transformation of E. coli cells is substantially enhanced by the method described by Hanahan (JMB (1983), hereinafter referred to as “Hanahan (1983)”). In the Hanahan (1983) method, the cells are grown at 37° C. in the presence of 20 mM Mg. Plasmid DNA is combined with the cells at 0° C. in the presence of Mn, Ca, Rb or K, dimethylsulfoxide (DMSO), dithiothreitol (DTT) and hexamine cobalt chloride. Competent cells of several strains of Escherichia coli (E. coli) prepared by the latter method have transformation efficiencies of from 1 to 5×108 transformants/μg plasmid DNA.
Another method for the preparation of competent E. coli cells is disclosed in U.S. Pat. No. 4,981,797 (Jessee and Bloom, 1991). That method includes the steps of growing the cells in a growth-conducive medium at a temperature of less than 37° C., rendering the cells competent and then freezing them.
Generally, frozen competent cells prepared by methods such as those summarized above have transformation efficiencies of about 1×108 transformants/μg plasmid DNA. These competent cells can be stored at −80° C. for several months without significant loss of transformation efficiency. However, cells prepared by the methods outlined above are extremely unstable when stored at temperatures higher than −80° C. (e.g., −20° C.). Stable storage of competent cells at higher temperatures is highly desirable, since many research labs do not have access to −80° C. freezers.